5-substituted picolinic acid compounds and their method of use

ABSTRACT

The present invention provides novel 5-substituted picolinic acid compounds of formula (I) or a pharmaceutically acceptable salt thereof:                    
     wherein R 1  and R 2  are independently H, C 2 -C 6  acyl or halo-substituted benzoyl; and R 3  is —C(O)O—C 1 -C 6  alkyl, C(O)OH, CN, CONH 2 , CONHCH 3 , CON(CH 3 ) 2 , 1-methyltetrazole or 2-methyltetrazole, with the proviso that when R 2  is acetyl and R 3  is methoxycarbonyl, R 1  is not H; and that when R 3  is CN, CONH 2 , CONHCH 3 , CON(CH 3 ) 2 , 1-methyltetrazole or 2-methyltetrazole, R 1  and R 2  are H. 
     The present invention also relates to a pharmaceutical composition comprising compound of the present invention, which is useful in the treatment of IL-1 and TNF mediated diseases or the like. 
     The present invention further relates to a process for producing the compounds of the formula (I).

This application is a division of U.S. application Ser. No. 09/178,949,filed on Oct. 26, 1998, entitled 5-Substituted Picolinic Acid Compoundsand their Production Process now U.S. Pat. No. 6,034,107, Mar. 7, 2000which is the national stage under 35 U.S.C. §371(a) of copendingInternational Patent Application Number PCT/IB97/01383, filed Nov. 4,1997.

TECHNICAL FIELD

This invention relates to novel 5-substituted picolinic acid compounds,and particularly to novel 5-substituted picolinic acid compoundsproduced by fermentation of a fungus Marasmiellus sp., which has beendeposited as FERM BP-5735. This invention also relates to processes forproducing the 5-substituted picolinic acid compounds, and apharmaceutical composition comprising the same, which is useful in thetreatment of IL-1 and TNF mediated diseases.

BACKGROUND ART

Interleukin-1 (IL-1) and tumor necrosis factor (TNF) are biologicalsubstances produced by a variety of cells, such as monocytes ormacrophages. IL-1 and TNF have been demonstrated to mediate a variety ofbiological activities thought to be important in immunoregulation andother physiological conditions such as inflammation.

There are many disease states in which excessive or unregulated IL-1production is implicated in exacerbating and/or causing the disease.These include rheumatoid arthritis, osteoarthritis, endotoxemia and/ortoxic shock syndrome, other acute or chronic inflammatory disease statessuch as the inflammatory reaction induced by endotoxin or inflammatorybowel disease; tuberculosis, atherosclerosis, muscle degeneration,cachexia, psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis,gout, traumatic arthritis, rubella arthritis, and acute synovitis.Recent evidence also links IL-1 activity to diabetes (T. Mandrup-Poulsenet al., Allergy, 1985, 40, 424). The only IL-1 blocker available todayis the natural IL-1 receptor antagonist (IL-1RA), which is easilymetabolized in the bloodstream with a very short half-life (E. V.Granowitz et al., Cytokine, 1992, 4, 353). Thus, active research hasbeen carried out to develop stable, long-acting agents which can betaken by oral administration or by parenteral injections rather than byintravenous infusion, which is required for IL-1RA. A number ofcompounds as IL-1 receptor antagonists, IL-1 biosynthesis inhibitors,and IL-1 converting enzyme inhibitors have been known (C. C. George etal., Exp. Opin. Ther. Paten, 1996, 6 (1), 41).

Excessive or unregulated TNF production has also been implicated inmediating or exacerbating a number of diseases including rheumatoidarthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis andother arthritic conditions; sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, cerebral malaria, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfusioninjury, acquired immunodeficiency syndrome (AIDS), AIDS related complex(ARC), keloid formation, scar tissue formation, Crohn's disease,ulcerative colitis, or pyresis. Although significant progress indeveloping potent TNF modulators has been achieved through the use ofrecombinantly derived proteins including monoclonal antibodies andsoluble receptors, the development of biosynthesis inhibitors andreceptor antagonists has been less successful. Recently a number ofsmall molecule TNF modulators have been claimed. Most of them whichspecifically inhibit TNF production do so by increasing intracellularcyclic adenosine monophosphate (cAMP) which ultimately blocks TNF geneexpression (Y. KATAKAMI et al., Immunology, 1988, 64, 719). The mostimportant of these compounds are the rolipram and pentoxifylline-relatedphosphodiesterase IV (PDE IV) inhibitors which are being pursued by anumber of pharmaceutical companies (A. BADGER et al., Circul. Shock,1994, 44, 188). The ability of thalidomide to block TNF productioncontributes to its therapeutic properties in humans (E. P. SAMPAIO etal., J. Exp. Med, 1991, 73, 699). Recent studies suggest thatcell-associated TNF may be necessary for normal host defense mechanisms.This finding has added to the excitement concerning the identificationof a unique metalloproteinase enzyme which is responsible for theproteolytic processing of TNF. Inhibitors of matrixmetalloproteinase-related enzyme have appeared (K. M. MOHLER et al.,Nature, 1994, 370, 218).

Inhibitors of interleukin 1, 6 and 8 and TNF are described in PCTapplication US94/07969 which was published on Jan. 26, 1995. Theinhibitors of TNF are also described in PCT application US94/04950 whichwas published on Nov. 24, 1994. Substituted picolinic acid compoundshave been known to be produced by fungus. These include phenopicolinicacid (5-(4-hydroxylbenzyl)picolinic acid) (T. Nakamura et al., J.Antibiotics, 27:477-, 1975), fusaric acid (5-butylpicolinic acid)( H.Hidaka et al., J.Antibiotics, 22:228-, 1969), and fusarinolic acid (K.Steiner et al., Helv. Chim. Acta, 54:845-, 1971).

The object of the present invention is to provide novel 5-substitutedpicolinic acid compounds having an excellent activities for TNF and/orIL-1 biosynthesis inhibition and a pharmaceutically compositioncomprising the same. Another object is to provide processes forproducing the novel 5-substituted picolinic acid compounds.

BRIEF DISCLOSURE OF THE INVENTION

Accordingly, the present invention provides novel 5-substitutedpicolinic acid compounds of formula (I):

or a pharmaceutically acceptable salt thereof,

wherein R¹ and R² are independently H, C₂-C₆ acyl or halo-substitutedbenzoyl; and

R³ is —C(O)O—C₁-C₆ alkyl, C(O)OH, CN, CONH₂, CONHCH₃, CON(CH₃)₂,1-methyltetrazole or 2-methyltetrazole, with the proviso that when R² isacetyl and R³ is methoxycarbonyl, R¹ is not H; and that when R³ is CN,CONH₂, CONHCH₃, CON(CH₃)₂, 1-methyltetrazole or 2-methyltetrazole, R¹and R² are H.

Preferred compounds of this invention are those of formula (I) shownabove, wherein R³ is —C(O)O—C₁-C₆ alkyl or C(O)OH, with the proviso thatwhen R² is acetyl and R³ is methoxycarbonyl, R¹ is not H.

The present invention also provides a culture of Marasmiellus sp. FERMBP-5735, which is capable of producing the 5-substituted picolinic acidcompounds, especially those of formula (I) wherein wherein R¹ and R² areH, and R³ is methoxycarbonyl (methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate), or R¹ is acetyl, R² isH, and R³ is methoxycarbonyl (methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate).

Further, the present invention provides a process for producing the5-substituted picolinic acid compounds of formula (I), which comprisescultivating a microorganism having identifying characteristics ofMorasmiellus sp. , FERM BP-5735, or a mutant or recombinant formthereof.

The present invention further provides a process comprising additionalsteps of isolating the 5-substituted picolinic acid compounds from thefermentation broth and chemically modifying the isolated compounds.

Also, the present invention provides a pharmaceutical composition foruse in the treatment of IL-1 and/or TNF mediated diseases, whichcomprises the 5-substituted picolinic acid compounds of formula (I)wherein R¹ and R² are H; and R³ is methoxycarbonyl (methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate); R¹ is acetyl; R² is H;and R³ is methoxycarbonyl (methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate); R¹ and R² are H;and R³ is C(O)OH (5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid); orR¹ and R² are acetyl; and R³ is methoxycarbonyl (methyl5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate); or pharmaceuticallyacceptable salt thereof in an amount effective in such treatments, and apharmaceutically acceptable carrier.

Also, the present invention provides a method for the treatment of IL-1and/or TNF mediated diseases, which comprises administering to saidsubject an antiinflammation amount of the compound of formula (I)wherein R¹ and R² are H; and R³ is methoxycarbonyl (methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate); R¹ is acetyl; R² is H;and R³ is methoxycarbonyl (methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate); R¹ and R² are H;and R³ is C(O)OH (5-(1,2-hydroxypropyl)-2-pyridinecarboxylic acid); orR¹ and R² are acetyl; and R³ is methoxycarbonyl (methyl5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate); and a pharmaceuticallyacceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The microorganism used in this invention is a strain of Marasmiellus sp.which was identified by and obtained from University of Tennessee. Itwas deposited under the accession number FERM BP-5735 to NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology (located at 1-3 Higashi 1-chome, Tsukuba, Ibaraki305, Japan) under the Budapest Treaty on Oct. 29, 1996. The taxonomicalproperties of the genus Marasmiellus have been reported by Singer, R.(in The Agaricales in modern taxonomy, 320-328, 1986).

In this invention, a mutant or recombinant form of FERM BP-5735 havingthe ability to produce the novel 5-substituted picolinic acid compoundsof formula (I) can be also used. The mutant or recombinant form may beobtained by spontaneous mutation, artificial mutation with ultravioletradiation, or treatment with mutagen such asN-methyl-N′-nitro-N-nitrosoguanidine or ethyl methanesulfonate, or acell technology method such as protoplast fusion, gene manipulation orthe like, according to well-known methods.

According to the present invention, the novel 5-substituted picolinicacid compounds of formula (I) may be produced by aerobic fermentation ofFERM BP-5735, or a mutant or recombinant form thereof, under conditionssimilar to those generally employed to produce bioactive compounds byfermentation.

FERM BP-5735, or a mutant or recombinant form thereof, is usuallyfermented under submerged aerobic conditions with agitation at atemperature of 20 to 40° C. for 1 to 20 days, which may be variedaccording to fermentation conditions. Cultivation of FERM BP-5735 toproduce said 5-substituted picolinic acid compounds of formula (I)preferably takes place in aqueous nutrient media at a temperature of 25to 35° C. for 10 to 15 days. The pH of medium may be adjusted in therange from 4.0 to 9.0, preferably from 5.5 to 7.0.

Nutrient media useful for fermentation include a source of assimilablecarbon such as sugars, starches and glycerol; a source of organicnitrogen such as casein, enzymatic digest of casein, soybean meal,cotton seed meal, peanut meal, wheat gluten, soy flour, meat extract andfish meal; a source of growth substances such as mineral salts, sodiumchloride and calcium carbonate; and trace elements such as iron,magnesium, copper, zinc, cobalt and manganese. If excessive foaming isencountered during fermentation, antifoam agents such as polypropyleneglycols or silicones may be added to the fermentation medium.

Aeration of the medium in fermentors for submerged growth is maintainedat 10 to 200%, preferably at 50 to 150% volumes of sterile air pervolume of the medium per minutes. The rate of agitation depends on thetype of agitator employed. A shake flask is usually run at 150 to 250rpm whereas a fermentor is usually run at 300 to 2,000 rpm.. Asepticconditions must, of course, be maintained through the transfer of theorganism and throughout its growth.

The 5-substituted picolinic acid compounds thus produced may be isolatedby standard techniques such as extraction and various chromatographictechniques.

As 5-substituted picolinic acid compounds of this invention, a compoundof formula (I) wherein R¹ and R² are H, and R³ is methoxycarbonyl(methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate); and a compoundof formula (I) wherein R¹ is acetyl, R² is H, and R³ is methoxycarbonyl(methyl 5-(1-acetoxy-2-hydroxypropyl)2-pyridinecarboxylate) wereisolated in a substantially pure form from the fermentation mixture. As5-substituted picolinic acid compounds of this invention, there weresynthesized 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid , methyl5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate, methyl5-(1,2-di-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate,5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide,5-(1,2-dihydroxypropyl)-N,N-dimethyl-2-pyridinecarboxamide,5-(1,2-dihydroxypropyl)-2-pyridinecarbonitrile,5-(1,2dihydroxypropyl)-2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridine and5-(1,2-dihydroxypropyl)-2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridinefrom methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate by chemicalmodification. These compounds were identified by various spectroscopictechniques such as UV spectrophotometry, NMR and-mass spectrometries,and the results will be shown in the section for working examples.

The compounds of formula (I) wherein R¹ and R² are an acyl group can beprepared by acylation of methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate, and the compounds offormula (I) wherein R³ is alkoxycarbonyl can be prepared by alkylationof demethyl methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate usingsuitable acylation and alkylating agents under suitable conditions knownto those skilled in the art.

Preferred compounds of this invention include those of formula (I),wherein

R¹ and R² are H; and R³ is methoxycarbonyl (methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate);

R¹ is acetyl; R² is H; and R³ is methoxycarbonyl (methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate);

R¹ and R² are H; and R³ is C(O)OH(5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid);

R¹ and R² are acetyl; and R³ is methoxycarbonyl (methyl5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate); and

R¹ and R² are p-bromobenzoyl; and R³ is methoxycarbonyl (methyl5-(1,2-di-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate).

Preferred compounds of this invention also include those of the formula(I), wherein

R¹ and R² are H, and R³ isCN(5-(1,2-dihydroxypropyl)-2-pyridinecarbonitrile);

R¹ and R² are H, and R³ is CONH₂(5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide);

R¹ and R² are H, and R³ is CONHCH₃(5-(1,2-dihydroxypropyl)-N-methyl-2-pyridinecarboxamide);

R¹ and R² are H, and R³ is CON(CH₃)₂(5-(1,2-dihydroxypropyl)-N,N-dimethyl-2-pyridinecarboxamide);

R¹ and R² are H, and R³ is 1-methyltetrazole(5-(1,2-di-hydroxypropyl)-2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridine);and

R¹ and R² are H, and R³ is 2-methyltetrazole(5-(1,2-di-hydroxypropyl)-2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridine).

More preferred compounds include a compound of formula (I), wherein R¹and R² are H; and R³ is methoxycarbonyl (methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate);

R¹ is acetyl; R² is H; and R³ is methoxycarbonyl (methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate);

R¹ and R² are H; and R³ is C(O)OH(5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid); and

R¹ and R² are acetyl; and R³ is methoxycarbonyl (methyl5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate).

Further preferred compounds of this invention also include those offormula (I) wherein

R¹ and R² are H, R³ is CONH₂(5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide)

R¹ and R² are H, R³ is CON(CH₃)₂(5-(1,2-dihydroxypropyl)-N,N-dimethyl-2-pyridinecarboxamide)

R¹ and R² are H, R³ isCN(5-(1,2-dihydroxypropyl)-2-pyridinecarbonitrile)

R¹ and R² are H, R³ is 1-methyltetrazole(5-(1,2-di-hydroxypropyl)-2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridine)and

R¹ and R² are H, R³ is 2-methyltetrazole(5-(1,2-di-hydroxypropyl)-2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridine).

The IL-1 and TNF biosynthesis inhibitory activities of theabove-mentioned 5-substituted picolinic acid compounds, methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate, (methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate,5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid , methyl5-(1,2-diacetoxypropyl)2-pyridinecarboxylate, methyl5-(1,2-di-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate,5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide,5-(1,2-dihydroxypropyl)-N,N-dimethyl-2-pyridinecarboxamide,5-(1,2-dihydroxypropyl)-2-pyridinecarbonitrile,5-(1,2-di-hydroxypropyl)-2-(2-methyl-2H-1,2,3,4tetrazol-5-yl)pyridineand5-(1,2-di-hydroxypropyl)-2-(1-methyl-1H-1,2,3,4tetrazol-5-yl)pyridine),were measured by the standard in vitro protocol described below. Thesecompounds were found to have the IL-1 and TNF biosynthesis inhibitoryactivities.

TNF bioassay

Heparinised human whole blood diluted four-fold with RPMI medium wasincubated with 10 μg/ml of Lipopolysaccharide (LPS) in the presence ofvarious concentrations of samples at 37° C. in a humidified atmospherecontaining 5% CO₂ for 4 h. The TNF titer in the supernatants wasdetermined with L929 cells which were destroyed by TNF quantitatively.L929 cells (2.5×10⁴ cells) in 90 μl of E-MEM medium containing 1% fetalcalf serum and 0.5 μg/ml of actinomycin D were placed in wells of96-well microplates (flat-bottom). Ten μl of the supernatants was addedto each well and incubated at 37° C. in a humidified atmospherecontaining 5% CO₂. After 18 h, the plates were rinsed with 0.9% sterilesaline and stained for 10 min with 0.4% crystal violet in MeOH. Theplates were washed with distilled water and were dried by air. Fifty μlof methanol was added to each well to elute the crystal violet, and theplates were read on a microplate reader (model 3550, BIO-RAD) at 595 nm.TNF production inhibitory activity is calculated by the formula:${{Inhibition}\quad (\%)} = {\left\{ {1 - \frac{\left\lbrack {{A_{595}\quad {Sample}} - {A_{595}\quad {Blank}}} \right\rbrack}{\left\lbrack {{A_{595}{Control}} - {A_{595}\quad {Blank}}} \right\rbrack}} \right\} \times 100}$

IL-1 bioassay

The supernatants prepared by the same method as TNF bioassay wereanalyzed IL-1 titer by commercially available specific ELISA system. Theplates were read on a microplate reader (model 3550, BIO-RAD) at 490 nm.IL-1 production inhibitory activity is calculated by the formula:${{Inhibition}\quad (\%)} = {\left\{ {1 - \frac{\left\lbrack {{A_{490}\quad {Sample}} - {A_{490}\quad {Blank}}} \right\rbrack}{\left\lbrack {{A_{490}{Control}} - {A_{490}\quad {Blank}}} \right\rbrack}} \right\} \times 100}$

The pharmaceutically acceptable salts of methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate, methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate,5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid, methyl5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate and methyl5-(1,2-di-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate are prepared ina conventional manner by treating a solution or suspension of thecompound with about one chemical equivalent of a pharmaceuticallyacceptable acid. Conventional concentration and recrystallizationtechniques are employed in isolating the salts.

Administration

The 5-substituted picolinic acid compounds of formula (I) apharmaceutically acceptable salt are useful in the treatment ofinflammation or the like. The 5-substituted picolinic acid compounds offormula (I) and a pharmaceutically acceptable salt may be administeredalone or in combination with pharmaceutically acceptable carriers, ineither single or multiple doses. Suitable pharmaceutical carriersinclude inert solid diluents or fillers, sterile aqueous solution andvarious organic solvents. The pharmaceutical compositions formed bycombining the 5-substituted picolinic acid compounds of formula (I) andthe pharmaceutically acceptable carriers are then readily administeredin a variety of dosage forms such as tablets, powders, lozenges, syrups,injectable solutions and the like. These pharmaceutical compositionscan, if desired, contain additional ingredients such as flavorings,binders, excipients and the like. Thus, for purposes of oraladministration, tablets containing various excipients such as sodiumcitrate, calcium carbonate and calcium phosphate may be employed alongwith various disintegrants such as starch, alginic acid and certaincomplex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often useful for tabletting purposes. Solid compositions of asimilar type may also be employed as fillers in soft and hard filledgelatin capsules. Preferred materials for this include lactose or milksugar and high molecular weight polyethylene glycols. When aqueoussuspensions or elixirs are desired for oral administration, theessential active ingredients therein may be combined with varioussweetening or flavoring agents, coloring matter or dyes and, if desired,emulsifying or suspending agents, together with diluents such as water,ethanol, propylene glycol, glycerin and combinations thereof.

For parenteral administration, solutions of the 5-substituted picolinicacid compounds of formula (I) and a pharmaceutically acceptable salt insesame or peanut oil, aqueous propylene glycol, or in sterile aqueoussolution may be employed. Such aqueous solutions should be suitablebuffered if necessary and the liquid diluent first rendered isotonicwith sufficient saline or glucose. These particular aqueous solutionsare especially suitable for intravenous, intramuscular, subcutaneous andintraperitioneal administration. In this connection, the sterile aqueousmedia employed are all readily available by standard techniques known tothose skilled in the art.

Additionally, the 5-substituted picolinic acid compounds of formula (I)and a pharmaceutically acceptable salt may be administered topicallywhen treating conditions of the skin and this may be done by way ofcreams, jellies, gels, pastes, and ointments, in accordance withstandard pharmaceutical practice.

In general, the 5-substituted picolinic acid compounds of formula (I) orits pharmaceutically acceptable salt are present in the above dosageforms at concentration levels ranging 5 to 70% by weight, preferably 10to 50% by weight.

In general, a therapeutically effective daily dose for the activecompound will range from 0.01 to 100 mg/kg, generally from about 1 toabout 5 mg/kg As is generally known, the effective dosage for the activecompound depends on the intended route of administration and otherfactors such as age and weight of the patient, as generally known to aphysician. The dosage also depends on the illness to be treated.

EXAMPLES

The present invention is illustrated by the following examples. However,it should be understood that the invention is not limited to thespecific details of these examples. Spectral and physico-chemical datawere obtained on the following instruments: IR, Shimadzu IR-470; UV,JASCO Ubest-30; Optical rotations, JASCO DIP-370 with a 5 cm cell; NMR,JEOL JNM-GX270 equipped with a LSI-11/73 host computer, TH-5 tunableprobe and version 1.6 software; El-MS, Hitachi M-80 with a direct inletmodule; and FAB-MS, JEOL JMS-700. The peak shapes are denoted asfollows: s (singlet), d (doublet), t (triplet), q (quartet), m(multiplet) and br (broad). FAB-MS spectra were measured using glycerolmatrix.

Example One Fermentation of Marasmiellus sp. (FERM BP-5735)

Cells from a 10- to 21-day-old petri dish of Marasmiellus sp. FERMBP-5735 grown on malt agar medium (malt extract 2.5% and agar 1.5%) wereharvested and suspended in 2 ml sterile water. This suspension was usedto inoculate two 500-ml flasks containing 100 ml of Medium-1 (glucose2%, malt extract 2%, yeast extract 0.18%, maltose 0.24% and agar 0.1%).The flasks were shaken at 26° C. for 7 days on a rotary shaker with 7-cmthrow at 220 rpm, to obtain a seed culture. The seed culture was used toinoculate forty 500-ml flasks containing 100 ml of Medium-2 (potatodextrose broth 2.4%). These flasks were shaken at 26° C. for 14 days ona rotary shaker with 7-cm throw at 250 rpm.

Extraction and Isolation

The fermentation broth (3 l) was filtered after the addition of 2 l ofethanol. The filtrate was concentrated to aqueous solution (1 l), whichwas then extracted 3 times with each of 1 l of n-butanol. The extractwas evaporated to afford an oily residue. The oily residue (3.5 g) wasapplied to a Shephadex LH-20 column (40×500 mm, Pharmacia trademark) andeluted with methanol. The active fractions applied to a YMC-pack ODSAM-343 column (20×250 mm, Yamamura trademark) and eluted withmethanol—water (15:85 to 70:30) for 45 min at a flow rate of 10 ml/min.The detection was made by UV absorbance at 220 nm. The eluted peaksshowing activity were collected to yield the compounds methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate (76.7 mg) and methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate (10.2 mg).

HPLC Analysis

Analytical HPLC of methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylateand methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate wasperformed using a YMC-pack ODS AM-312 column (6.0×150 mm, Yamamuratrademark) and eluted with methanol—water (20:80 to 70:30) for 10 minand continuatively to MeOH for 5 min at a flow rate of 0.8 ml/min. Theretention times (min) were methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate (7.7) and methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate (10.9).

Characterization

The physico-chemical properties and spectral data of methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate and methyl5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate were as follows:

methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate: white amorphouspowder; molecular formula C₁₀H₁₃NO₄; LRFAB-MS m/z 212 [M+H]⁺; HRFAB-MSm/z 212.0940 (calcd. for C₁₀H₁₄NO₄, 212.0923); [α]_(D) ²⁴ +20.0° (c0.13, MeOH); UV 1_(max) (MeOH) nm (e) 230 (9,500), 270 (5,800); IRγ_(max) (KBr) cm⁻¹ 3325, 1736, 1437, 1309, 1257, 1122, 1089, 1028, 1001,817, 709; ¹H NMR (CD₃OD) δ 8.66 (1H, d, J=1.9Hz), 8.12 (1H, d, J=8.1Hz), 7.99 (1 H, dd, J=8.1 and 1.9 Hz), 4.55 (1H, d, J=5.9 Hz), 3.96 (3H,s), 3.86 (1H, dq, J=6.2 and 5.9 Hz), 1.17 (3H, d, J=6.2 Hz); ¹³C NMR(CD₃OD) d 167.39 (s), 150.60 (d), 148.20 (s), 144.80 (s), 138.57 (d),126.57 (d), 77.49 (d), 72.79 (d), 53.95 (q), 19.95 (q).

methyl 5-(1-acetoxy-2-hydroxypropyl)-2-pyridinecarboxylate: whiteamorphous powder; molecular formula C₁₂H₁₅NO₅; LRFAB-MS m/z 254 [M+H]⁺;HRFAB-MS m/z 254.1051 (calcd. for C₁₂H₁₆NO₅, 254.1028); [α]_(D) ²⁴+27.1° (c 0.17, MeOH); UV 1_(max) (MeOH) nm (e) 230 (8,200), 270(4,400); IR γ_(max) (KBr) cm⁻¹ 3465, 1732, 1435, 1370, 1309, 1239, 1120,1058, 813, 785, 712; ¹H-NMR (CD₃OD) δ 8.67 (1H, d, J=1.6 Hz), 8.13 (1 H,d,J=8.1 Hz), 8.01 (1H, dd,J=8.1 and 1.6 Hz), 5.03 (1H, dt,J=6.2 and 5.4Hz), 4.82 (1H, d, J=5.4 Hz), 3.97 (3H, s), 1.95 (3H, s), 1.20 (3H, d,J=6.2 Hz).

Example Two Preparation of 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylicacid

To a solution of methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate (5mg) in water (0.1 ml), 1N lithium hydroxide (50 μl) was added at roomtemperature. After stirring for 1 hour at room temperature, the reactionmixture was neutrized with 1N HCl. The solution was applied to a DiaionHP20 (Mitsubishikasei trademark) column and eluted with methanol-water(1:1) to give 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylic acid: whiteamorphous powder; molecular formula C₉H₁₁NO₄; LRFAB-MS m/z 196 [M−H]⁻;¹H-NMR (D₃O) δ 8.74 (1 H, d, J=2.2 Hz),8.47(1 H,d, J=8.1Hz),8.28(1H,dd,J=8.1 and2.2 Hz),4.88(1 H,d,J=4.3 Hz), 4.12 (1 H, dq,J=6.5 and 4.3 Hz), 1.21 (3H, d, J=6.5 Hz).

Example Three Preparation of methyl5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate

To a solution of methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate (6mg) in pyridine (0.1 ml), acetic anhydride (50 μl) was added at roomtemperature. After stirring for 1 hour at room temperature, the reactionmixture was evaporated under N₂ gas. The residue was applied to a silicagel plate (Kiesselgel GF₂₅₄, 10×10 cm, Merck trademark) and developedwith chloroform-methanol (95:5) to give methyl5-(1,2-diacetoxypropyl)-2-pyridinecarboxylate: white amorphous powder;molecular formula C₁₄H₁₇NO₆; LRFAB-MS m/z 296 [M+H]⁺; ¹H-NMR (CDCl₃) δ8.68 (1 H, d, J=2.2 Hz), 8.16 (1H, d, J=8.1 Hz), 8.03 (1 H, dd, J=8.1and 2.2 Hz), 5.95 (1 H, d, J=4.3 Hz), 5.28 (1H, dq, J=6.5 and 4.3 Hz),3.97 (3H, s), 2.12 (3H, s), 1.99 (3H, s), 1.18 (3H, d, J=6.5 Hz).

Example Four Preparation of methyl5-(1,2-di-bromobenzoyloxypropyl)-2-pyridinecarboxylate

To a solution of methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate(4.1 mg) and a catalic amount of 4-N,N-dimethylaminopyridine in pyridine(1 ml), p-bromobenzoyl chloride (10 mg) was added at room temperature.After stirring at 90° C. for 3 days, the reaction mixture was evaporatedunder N₂ gas. The residue was applied to a silica gel plate (KiesselgelGF₂₅₄, 10×10 cm, Merck trademark) and developed with chloroform-methanol(95:5) to give 1.03 mg of methyl5-(1,2-di-p-bromobenzoyloxypropyl)-2-pyridinecarboxylate: whiteamorphous powder; molecular formula C₂₄H₁₉Br₂NO₆; LREI-MS m/z 577 [M]⁺;¹H-NMR (CDCl₃) δ8.88 (1H, d, J=2.0 Hz), 8.16 (1 H, d, J=8.4 Hz), 7.94 (1H, dd, J=8.4 and 2.2 Hz), 7.89 (2H, d, J=8.4 Hz), 7.80 (2H, d, J=8.4Hz), 7.61 (2H, d, J=8.4 Hz), 7.58 (2H, d, J=8.4 Hz), 6.27 (1 H, d, J=4.4Hz), 5.68 (1H, dq, J=6.6 and 4.4 Hz), 4.01 (3H, s), 1.41 (3H, d, J=6.6Hz).

Example Five Preparation of 5-(1,2-dihydroxpropyl)-2-pyridinecarboxamide

A homogeneous mixture of methyl5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate (70.0 mg, 0.33 mmol) and a2.0M solution of ammonia in methanol (Aldrich, 15.0 ml, 30.0 mmol) wasstirred and heated at a bath temperature between 50 and 60° C. in amicroreactor overnight. After cooling, the reaction mixture wasconcentrated in vacuo to give a white solid (64.0 mg). This was purifiedby preparative TLC [Merck Kieselgel 60, Art 1.05744, 0.5 mm thick, ×2;development: CH₂Cl₂-MeOH (8:1); elution: CH₂Cl₂-MOH (3:1), 240 ml]. Therecovered white solid residue was suspended in THF, and the mixture wasfiltered through a short pad of Celite. The filter cake was washed withTHF. The combined filtrate and washings were concentrated in vacuo togive 5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide as a white solid(70.7 mg, quantitative): ¹H-NMR (270 MHz) δ(CDCl₃+DMS0-d₆) 8.57 (d,J=2.2 Hz, 1 H), 8.11 (d, J=8.1 Hz, 1H), 7.89 (dd, J=8.1, 2.1 Hz, 1H),7.86 (br.s, 1 H), 6.71 (br.s, 1 H), 4.98 (d, J=4.3 Hz, 1 H), 4.67 (dd,J=4.3, 4.3Hz, 1 H), 4.00 (d, J=5.1 Hz, 1 H), 4.00˜3.87 (m, 1 H), 1.07(d, J=6.2 Hz, 3H) ppm; MS m/z 197 (0.83%, M⁺1), 152 (100%).

Example Six Preparation of5-(1,2-dihydroxypropyl)-N,N-dimethyl-2-pyridinecarboxamide

A mixture of methyl 5-(1,2-dihydroxypropyl)-2-pyridinecarboxylate (52.0mg, 0.25 mmol) and a 2.0M solution of dimethylamine in methanol(Aldrich, 15.0 ml, 30.0 mmol) was stirred and heated at a bathtemperature of 100° C. in a microreactor for four nights. After cooling,the reaction mixture was concentrated in vacuo. The residue (60.3 mg)was purified by preparative TLC [Merck Kieselgel 60, Art 1.05744, 0.5 mmthick, ×2; development: CH₂Cl₂ - MeOH (8:1); elution: CH₂Cl₂ - MOH(3:1), 240 ml]. The recovered white solid residue was suspended in THF,and the mixture was filtered through a short pad of Celite. The filtercake was washed with THF. The combined filtrate and washings wereconcentrated in vacuo to give5-(1,2-dihydroxypropyl)-N,N-dimethyl-2-pyridinecarboxamide as a whitesolid (45.2 mg, 80.6%): ¹H-NMR (270 MHz) δ(CDCl₃): 8.41 (d, J=1.8 Hz, 1H), 7.76 (dd, J=7.9, 1.8 Hz, 1H), 7.52 (d, J=7.9 Hz, 1 H), 4.86 (d,J=3.7 Hz, 1 H), 4.11˜3.97 (m, 1 H), 3.80 (br.s, 1H), 3.13 (s, 3H), 3.04(s, 3H), 2.68 (br.s, 1H), 1.15 (d, J=6.6 Hz, 3H) ppm; MS m/z: 225 (7.9%,M⁺+1), 224 (50.9%, M⁺), 153 (100%).

Example Seven Preparation of5-(1,2-dihydroxypropyl)-2-pyridinecarbonitrile

To a solution of 5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide (29.5 mg,0.142 mmol) in DMF (2 ml) were added 2-methoxypropene (41 ml, 0.425mmol) and p-toluenesulfonic acid monohydrate (4.9 mg, 0.0283 mmol) atroom temperature. After stirring at room temperature for 70 min, themixture was basified with NaHCO₃. The mixture was diluted with ethylacetate (30 ml), washed with water (20 ml ×2), and dried over Na₂SO₄.After the solvent was evaporated in vacuo, the oily residue was purifiedby preparative TLC [acetone/hexane (1/2, v/v)] to afford 16.4 mg (49%)of 5-(1,2-dihydroxypropyl)-2-pyridinecarboxamide as a white solid:¹H-NMR (270 MHz, CDCl₃) δ 8.49 (1H, d, J=1.8 Hz), 8.20 (1 H, d, J=8.1Hz); 7.84 (1 H, br s), 7.80 (1H, dd, J=1.8 and 8.1 Hz), 5.89 (1 H, brs), 5.27 (1 H, d, J=7.0 Hz), 4.66 (1 H, quint, J=6.5 Hz), 1.66 and 1.49(each 3 H, 2 s), 0.82 (3 H, d, J=6.5 Hz) ppm.

To a stirred solution of5-(2,2,5-trimethyl-1,3-dioxolan-4-yl)-2-pyridinecarboxamide (16.4 mg,0.0695 mmol) in 1,4-dioxane (1 ml) were added pyridine (23 ml, 0.278mmol) and trifluoroacetic anhydride (20 ml, 0.139 mmol) at roomtemperature. After stirring at room temperature for 1 h, the mixture wasdiluted with ethyl acetate (20 ml), washed with sat. NaHCO₃ (10 ml ×2),dried over Na₂SO₄, and concentrated in vacuo to give a brown syrup. Thiswas purified by preparative TLC [acetone/hexane (1/4, v/v)] to afford9.7 mg (64%) of5-(2,2,5-trimethyl-1,3-dioxolan-4-yl)-2-pyridinecarbonitrile as asolid:¹H-NMR (270 MHz, CDCl₃) δ 8.62 (1H, d, J=1.8 Hz), 7.80 (1H, dd,J=1.8 and 8.1 Hz), 7.70 (1H, d, J=8.1 Hz), 5.25 (1H, d, J=7.0 Hz), 4.67(1H, quit. J=6.6 Hz), 1.64 and 1.48 (each 3H, 2 s), 0.82 (3 H, d, J=6.6Hz) ppm.

A mixture of (3) (9.7 mg, 0.0444 mmol) and 80% aq. acetic acid (2 ml)was stirred and heated at 60° C. for 2.5 h. The mixture was concentratedin vacuo to give a syrup. This was purified by preparative TLC[methanol/dichloromethane (1/10)] to afford 7.4 mg (94%) of5-(1,2-dihydroxypropyl)-2-pyridinecarbonitrile: ¹H-NMR (270 MHz, CDCl₃)δ 8.68 (1H, d, J=2.2 Hz), 7.91(1H, dd, J=2.2 and 8.1 Hz), 7.70 (1H, d,J=8.1 Hz), 4.84 (1H, d, J=3.7 Hz), 4.11 (1H, dt, J=6.2, 6.2, and 10.3Hz), 2.99 (1H, br s), 2.31 (1H, br s), 1.06 (3H, d, J=6.2 Hz) ppm.

Example Eight Preparation of5-(1,2-di-hydroxypropyl)-2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridineand 5-(1,2-di-hydroxypropyl2(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridine

To a solution of 5-(1,2 dihydroxypropyl)-2-pyridinecarboxamide (161 mg,0.771 mmol) in DMF (6 ml) were added imidazole (1.05 g, 15.4 mmol) andt-butylchlorodimethylsilane (1.16 g, 7.71 mmol) at room temperature.After the mixture was stirred and heated at 70° C. for 5.5 h, water (2ml) was added, and the stirring was continued under the same heatingconditions for 2 h. The mixture was diluted with ethyl acetate (200 ml),washed with water (100 ml ×4), dried over Na₂SO₄, and concentrated invacuo to give a crystalline residue. This was chromatographed on silicagel (20 g). Elution with ethyl acetate/hexane (1/4, v/v) afforded 326 mg(99%) of5-[1,2-di-{(1(tert-butyl)-1,1-dimethylsilyloxy}propyl]-2-pyridinecarboxamideas a white solid: ¹H-NMR (270 MHz, CDCl₃) δ 8.50 (1H, d, J=1.8 Hz), 8.15(1 H, d, J=8.1 Hz), 7.86 (1 H, br s), 7.81 (1 H, dd, J=2.2 and 8.1 Hz),5.89 (1 H, be s), 4.40 (1 H, d, J=6.6 Hz), 3.74 (1 H, quint., J=6.1 Hz),1.23(3 H, d, J=6.2 Hz), 0.87 and 0.75 (each 9 H, 2 s), 0.06, −0.11,−0.18, and −0.40 (each 3H, 4 s) pm.

To a stirred solution of5-[1,2-di-{(1-(tert-butyl)-1,1-dimethylsilyloxy}propyl]-2-pyridinecarboxamide(326 mg, 0.769 mmol) in 1,4-dioxane (8 ml) were added pyridine (0.25 ml,3.07 mmol) and trifluoroacetic anhydride (0.22 ml, 1.54 mmol) at roomtemperature. After stirring at room temperature for 0.5 h, the mixturewas diluted with ethyl acetate (100 ml), washed with sat. NaHCO₃ (50 ml×2), dried over Na₂SO₄, and concentrated in vacuo to give an oilyresidue. This was chromatographed on silica gel (30 g). Elution withethyl acetate/hexane (1/15, v/v) afforded 264 mg of5-[1,2-di-{(1-(tert-butyl)-1,1-dimethylsilyloxy}propyl]-2-pyridinecarbonitrileas a solid: ¹H-NMR (270 MHz, CDCl₃) δ 8.65 (1 H, d, J=1.8 Hz), 7.80 (1H, dd, J=1.8 and 7.7 Hz), 7.65 (1 H, d, J=7.7 Hz), 4.39 (1 H, d, J=6.6Hz), 3.72 (1 H, quint, J=6.2 Hz), 1.22 (3 H, d, J=6.2 Hz), 0.88 and 0.75(each 9 H, 2 s), 0.07, −0.08, −0.18, and −0.38 (each 3 H, 4 s) ppm.

To a solution of5-[1,2-di-{(1-tert-butyl)-1,1-dimethylsilyloxy}propyl)-2-pyridinecarbonitrile(191 mg, 0.468 mmol) in toluene (4 ml) were added NaN₃ (122 mg, 1.87mmol) and tributyltin chloride (0.51 ml, 1.87 mmol) at room temperature.The stirring was continued with heating at reflux for 27 h After themixture was diluted with toluene (2 ml), 1M NaOH (2.4 ml) and MeI (0.6ml, 9.37 mmol) were added at room temperature. After stirring at roomtemperature for 3 h, the mixture was diluted with ethyl acetate (100ml), washed with water (50 ml ×3), dried over Na₂SO₄, and concentratedin-vacuo to give an oily residue. This was purified by preparative TLC[acetone/hexane (1/5, v/v)] to afford 149 mg (68%) of5-[1,2di-{(1-(tert-butyl)-1,1dimethylsilyloxy}propyl]-2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridine:¹H-NMR (270 MHz, CDCl₃) δ 8.64 (1 H, d, J=1.8 Hz), 8.31 (1 H, d, J=8.4Hz), 7.88 (1 H, dd, J=1.8 and 8.4 Hz), 4.58 (3 H, s), 4.42 (1 H, d,J=7.0 Hz), 3.77 (1 H, quint, J=6.2 Hz), 1.26 (3 H, d, J=6.2 Hz), 0.90(and 0.76 (each 9 H, 2 s), 0.09, −0.07, −0.15, and −0.37 (each 3 H, 4 s)ppm; and 63 mg (29%) of5-[1,2-di-{(1-(tert-butyl)-1,1-dimethylsilyloxy}propyl]-2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridine:¹H-NMR (270 MHz, CDCl₃) δ 8.72 (1 H, d, J=1.8 Hz), 8.19 (1 H, d, J=8.1Hz), 7.82 (1 H, dd, J=2.2 and 8.1 Hz), 4.45 (3 H, s), 4.41 (1 H, d,J=6.6 Hz), 3.79 (1 H, quint, J=6.2 Hz), 1.23 (3 H, d, J=6.2 Hz), 0.88and 0.75 (each 9 H, 2 s), 0.07, −0.09, −0.17, and 0.36 (each 3 H, 4 s)ppm.

To a solution of5-[1,2-di-{(1-tert-butyl)-1,1-dimethylsilyloxy}propyl]-2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridine(149 mg, 0.320-mmol) in THF (4 ml) were added acetic acid (73 ml, 1.28mmol) and 1M tetrabutylammoium fluoride (TBAF) (1.3 ml, 1.28 mmol) atroom temperature. After stirring for 2.5 h, the mixture was concentratedin vacuo to give a syrupy residue. This was purified by preparative TLC(acetone/hexane (1/1, v/v)] to give 42 mg (56%) of5-(1,2-di-hydroxypropyl)-2-(2-methyl-2H-1,2,3,4-tetrazol-5-yl)pyridine:¹H-NMR (270 MHz, CDCl₃) δ 8.68 (1 H, d, J=1.8 Hz), 8.19 (1 H, d, J=8.1Hz), 7.93 (1 H, dd, J=2.0 and 8.2 Hz), 4.85 (1 H, d, J=3.7 Hz), 4.47 (3H, s), 4.19-4.11 (1 H, m), 3.99 (1 H, br s), 3.27 (1 H, br s), 1.09 (3H, d, J=6.2 Hz) ppm.

To a solution of5-[1,2-di-{(1-(tert-butyl)-1,1-dimethylsilyloxy}propyl]-2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridine(76.1 mg, 0.164 mmol) in THF (3 ml) were added acetic acid (38 ml, 0.656mmol) and 1M TBAF (0.7 ml, 0.656 mmol) at room temperature. Afterstirring at room temperature for 2 h, the mixture was concentrated invacuo to give a syrupy residue. This was purified by preparative TLC[acetone/hexane (1/1, v/v)] to give 24 mg (63%) of5-(1,2-di-hydroxypropyl)-2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)pyridine:¹H-NMR (270 MHz, CDCl₃) δ 8.69 (1 H, d, J=2.0 Hz), 8.14 (1 H, d, J=8.1Hz), 7.89 (1 H, dd, J=2.0 and 8.1 Hz), 4.85 (1 H, d, J=3.7 Hz), 4.44 (3H, s), 4.17-4.08 (1 H, m), 3.83 (1 H, br s), 3.13 (1H, br s), 1.07 (3 H,d, J=6.2 Hz) ppm.

The chemical structure of the compounds prepared in the examples are inthe following Table.

TABLE (I)

Example Number R¹ R² R³ 1 H H CH₃—O—C(O)— 1 CH₃C(O)— H CH₃—O—C(O)— 2 H HCOOH 3 CH₃C(O)— CH₃C(O)— CH₃—O—C(O)— 4

CH₃—O—C(O)— 5 H H CONH₂ 6 H H CON(CH₃)₂ 7 H H CN 8 H H

8 H H

What is claimed is:
 1. A method for the treatment of IL-1 and/or TNFmediated diseases of a subject in need of such tretment, which comprisesadministering to said subject an anti-inflammatory amount of a compoundaccording to the formula (I):

wherein: a) R¹ and R² are H and R³ is methoxycarbonyl; b) R¹ is acetyl,R² is H and R³ is methoxycarbonyl; c) R¹ and R² are H and R³ is COOH, ord) R¹ and R² are acetyl and R³ is methoxycarbonyl.